The emerging field of synthetic genetics provides an exciting opportunity to explore the structural and functional properties of synthetic genetic polymers by in vitro selection. However, achieving the goal of artificial genetics requires the ability to synthesize unnatural nucleic acid substrates (“XNA”s), such as threose-nucleic acids (“TNAs”), that are not otherwise available. Limiting this process, however, is the availability of enzymes and conditions that allow for the storage and propagation of genetic information present in unnatural nucleic acid polymers such as TNAs.
Threose nucleic acid (TNA) is an unnatural genetic polymer composed of repeating units of a-L-threofuranosyl nucleic acids with 2′3′-phosphodiester linkages. In 2001, TNA was shown to form stable antiparallel Watson-crick duplexes with complementary strands of DNA and RNA, which provides a mechanism for exchanging genetic information with DNA and RNA. The structure of an all-TNA duplex, indicates that TNA is structurally similar to A-form RNA.
The discovery of TNA as an alternative genetic polymer with an RNA-like structure inspired other laboratories to begin developing the methodology needed to explore the functional properties of TNA by in vitro selection. Much of the early work in this area focused on identifying polymerases that could recognize TNA either in the template or as a nucleoside triphosphate. From these studies, we identified several DNA polymerases that could synthesize short sequences of DNA on a TNA template and other polymerases that could copy limited stretches of TNA on a DNA template. Herdewijn reported similar findings for the transcription of tTTP on a DNA template using thermophilic polymerases.
While these results show that TNA is not easily recognized by natural enzymes, subsequent screening did lead to the discovery of Therminator DNA polymerase, an engineered form of the Archeal family B replicative DNA polymerase isolated from the Thermococcus species 9° N. Relative to the natural enzyme, Therminator contains the mutations D141A and E143A in the exonuclease domain, as well as the mutation A485L in the finger domain. Therminator DNA polymerase was originally developed by NEB to improve the incorporation fluorescent nucleotides for Sanger sequencing. However, we found that under certain conditions, Therminator DNA polymerase functions can also function as an efficient DNA-dependent TNA polymerase.
Using Therminator DNA polymerase to synthesize TNA on a DNA template, Ichida and Szostak developed a DNA display strategy to generate functional TNA molecules by in vitro selection. This method establishes a genotype-phenotype link by extending a library of self-priming DNA templates with TNA, which allows each TNA sequence to become physically connected to its own DNA message. Using DNA display, we evolved a TNA aptamer with high affinity and high specificity to human thrombin. This demonstration showed that TNA can fold into tertiary structures with ligand binding activity.
In 2013, we developed a two-enzyme replication system for TNA that mimics the natural process of RNA transcription and reverse transcription. This approach was developed to expand the range of evolutionary strategies that could be used to evolve TNA aptamers and catalysts by in vitro selection. This system uses Therminator DNA polymerase to copy DNA into TNA and the Superscript II to copy TNA back into DNA. Using this strategy, TNA replication was limited to a three letter genetic alphabet due to problems associated with the incorporation of tCTP opposite G in the template. When G residues are present in the DNA template, we observe ˜30% G to C transversions in the replicated DNA (DNA to TNA to DNA), suggesting that G:G mispairing occurs during TNA synthesis.
The ability to convert genetic information back and fourth between DNA and TNA has an enormous impact on biotechnology, molecular medicine, and information storage. This technology could be used to make diagnostic and therapeutic molecules that are extremely resistant to nuclease degradation. It could also be used to store information in a biologically safe medium.